SU677666A1 - Method of producing 7l-or extradiols - Google Patents

Abstract

1528796 7α-Hydroxy-cotradiols SCHERING AG 13 Oct 1975 [14 Oct 1974 8 Aug 1975] 41798/75 Heading C2U Novel steroids of the formula (wherein R 1 is H, acyl, optionally substituted alkyl, cycloalkyl or a saturated oxygen-containing heterocyclic group; R 2 and R 3 are each H, acyl or a saturated oxygen-containing heterocyclic group; and R 4 is a C 1-5 aliphatic hydrocarbon group which may be chloro-substituted if it is unsaturated) are prepared from the corresponding 17-ones and suitable reagents containing R 4 . They may also be obtained by microbiological hydroxylation of the corresponding 7-unsubstituted-17#-ols. In the products OH groups may be esterified or etherified and esters and ethers may be hydrolysed. 3,7α - bis - tetrahydropyranyloxy - #<SP>1,3,5(10)</SP>- estratrien-17-one is prepared from the corresponding 3,7-diol and dihydropyran. 3-Cyclopentyloxy - 7α - hydroxy - #<SP>1,3,5(10)</SP>-estratrien - 17- one is prepared from the same starting material and cyclopentyl bromide. The novel steroids possess a strong vaginotropic and a weak uterotropic action and they are suitable for treatment of estrogen deficiency phenomena, particularly for postmenopausal treatment of women. They may be made up into pharmaceutical compositions with suitable carriers.

Description

ridbv or acid chlorides c. presence of catalysts. Tsvets

Lr (5Yuk S are drilled and cleaned by known methods.

Example 1. Microbiological 7-hydroxylation.

An Erlenmeyer flask with a capacity of 2 liters, 5 containing 500 ml of a nutrient solution containing,% sterilized for 30 minutes at 120 ° C in an autoclave, containing:% glucose 3; Corn steep 1 j; NaNO3 0.2, KH2POj 0.1; 0.2, MgSO 0.05; FeS04 0.002, and KCl 0.05, are inoculated with a lyophilic culture of biplodia hatalensis (ATCC 9055) and shaken for 72 hours while on a rotary shaker. This preliminary culture is then seeded with a fermenter with a capacity of 20 liters, which is filled with 15 liters sterilized at a pressure of 1.1 atm. The environment of that composition, which is also a preliminary culture.

With the addition of silicone SH as an antifoaming agent, they are germicated with, with aeration of 10 l / min, pressure of 0.7 atm and stirring. - 220 rpm for 24 hours 1 liter; culture broth under sterile conditions is transferred to 14 l sterilized

as above, the nutrient medium of such composition is grown with the same words. After 12 hours, a stably filtered solution is added; from 3 g of ethinyl estradiol in 100 ml of dimethyl sulfoxide.

The course of the transformation is followed

analysis with the use of thin-layer 35 rotography of methyl-butyl-ketone extracts of the fermenter samples. After complete conversion (45 hours of contact time), the contents of the enzyme are mixed with methylysobutyl ketone, taking 10 l each time, and the extract is evaporated in vacuo at bath temperature.

Residue To remove the antifoam, it is dissolved in methanol. Siggyc oil is separated in a separatory funnel, then the solution is filtered through a folded filter and the powder is evaporated to dryness. Remaining

The product is now dissolved in methylene chloride and graphened through a silica gel column using a gradient of methylene chloride / acetone for purification. After crystallization from acetic ether, pure 17c-ethynyl-1 3.5 / 10 / -estra-55triene-3.7s, white 17-triol melts at 234-235 ° C (decomposition). C, (MOS weight 312.4).

Example 2. 500 mg of 17-ethynyl-1, 3.5 / 10 / -estratriene-3.7 °, Q 17r -triol is dissolved in 10 ml of pyridine, 0.5 / HGH is addedGaTayTadrya and stirred for 20 minutes

Cool with ice. Then the solution is mixed into 100 ml of cooled 8%

sulfuric acid and the precipitation is washed to neutral r.eaktsii. After drying, it is recrystallized from a mixture of ether / hexane to obtain pure 3-acetoxy-17 C-ethgl-, 3.5 / 10 / estratrien-7 °, 17P-diol with mp 133335 ° C, 17 ° / -ethynyl- 1,3,5 / 10 / -estratrien-3, 7c (, 17B-triol with mp 232 CC;

Example 3. A solution of 100 mg of 17a-ethynyl-1,3,5 / 10 / -estratriene 3.7d, 17r -triol in 5 ml of ethyl acetate at room temperature is mixed successively with .1 ml of acetic anhydride and 1 drop of perchloric acid (70 %-Noah). After 3 minutes, 1 drop of pyridine was added, washed with saturated sodium chloride solution, dried and evaporated. 78 mg of 3.7 cc, 17/3-triacetoxy17 o6-ethynyl-1, 3.5- / 10 / -estratriene are obtained in the form of an amorphous substance. UV spectrum (methanol): -2 f9760; 2b-) 6267-715; e274 - b96.

Example4. To a solution of 150 mg 17c / -ethyn-1, 3.5 / 10 / -estratriene3, 7aLf 17- | -triol in 3 ml of pyridine was added 1 ml of acetic anhydride and left for 16 hours at room temperature. The composition, with the addition of cyclohexane or CC1d, was evaporated to dryness. The residue is dissolved in ethyl acetate and washed with water. After drying and evaporation, 120 ml of crude product are obtained, which is purified by thin layer chromatography. Obtain 110 mg of 3.7oC-diacetoxy-17 on ethinyl-, 3.5 / 10 / -estratrien-17 -ol, m.p. 165-16bS

EXAMPLE 5: 200 mg of 17 °; ethynyl-1, 3.5 / 10 / -estratriene-3.7, 17fb-triol is dissolved in 3 ml of meta-, nola and 1 ml of cyclopentyl bromide upon heating, then add 200 mg of potassium carbonate and kitts for 12 hours under a nitrogen atmosphere. Thereafter, the mixture is added to ice-acetate water and extracted with methylene chloride. The organic phase is washed to neutral, dried and evaporated. The crude product (150 mg) was purified by thin layer chromatography, and 100 mg of 17o ethynyl-3-cyclopent and Loxy-1, 3.5 / 10 / estratrien-7-6, 17P.-diol were obtained. M.p.126, 12.8 p.

PRI me R 6. A solution of 350 mg llyethenyl-3-cyclopentyloxy-1, 3.5 / 10 / estratrien-7o. , 17 / -diol in 5 ml of ethyl acetate is mixed with 1 ml of acetic anhydride and 1 drop of chloric acid (70%) and stirred for 3 minutes at room temperature. Then add 0.5 ml of pyridine, about .ivuaBaroT. saturated with sodium chloride solution, dried and evaporated. 310 mg of 7 ° C are obtained, 17p -diacetoxy17 oL-ethynyl-3-cyclopentyloxy-1,3,

5/10 / -estratriene in the form of an amorphous substance. UV spectrum (methanol); z2.2 9060; 277-8 7 30; 2: 1920; .

Example 7: 500 ml of 17 ° -ethyn1, 3, .5 / 10 / -estratriene-3, 70 /, 1 / 7/3 of the triol are dissolved in 10 ml of pyridine and 1 ml of butyric acid anhydride is added and stirred .60 min at room temperature. Then the solution is stirred into 100 ml of cooled 8% sulfuric acid and the precipitated precipitate is washed to neutrality. After drying, it is recrystallized from ether / hexane to give pure 3-butyloxy-17a. , ethinyl-1, 3, 5/10 / -estratrien-7, 17p-diol with m.p. 130-l3lc. .

Example 500 mg of 17 °.-Ethynyl-1, 3.5 (10) -estratriene-3.7 ° C, 17p-triol is dissolved in 10 ml of pyridine, 3 ml of oily anhydride are added and the mixture is stirred for 72 hours at room temperature. Then the solution is stirred into 100 ml of cooled 8% sulfuric acid and the precipitated precipitate is washed until neutral. For further purification, the product is chromatographed over a silica gel column and recrystallized from ether / hexane. Pure 3, IcL-dibutyryloxy-17o / - - ethynyl-1,3,5 / 10 / estratriene-17fc-ol melts at 132.5-133 ° C.

Sample To a solution of 450 mg of 3-acetoxy 17o-ethynyl-1, 3.5 / 10 / -estratriene-7 CU-, 17 US-diol in 30 ml of absolute benzene, 7 ml of triethylamine are added at room temperature and with vigorous stirring 1, 4 ml of isopropylsulfonic acid chloride and stirred for 48 hours at room temperature. The mixture is then poured onto ice and the substance is extracted with ether. The ether phase is washed, dried and evaporated and the crude product is purified using gradient siroathografii., Obtain 250 mg of 17 o-ethynyl-3-acetoxy-7 o-isopropylsulfo-yloxy-1, 3.5 / 10 / -estratriene-17.

I'll try it on. A solution of 350 mg of 17-ethynyl-1, 3.5 / 10 / -estratriene 3, 7 ° C, 17-triol in 35 ml of absolute benzene is mixed with 5 ml of triethylamine at room temperature, with vigorous stirring, and with 2 ml of isopropylsulfoxyloxy chloride. Allow to stir at room temperature for 38 hours, then poured onto ice and extracted with ether. The ether phase is washed with water, dried and evaporated. The crude product is purified by gradient chromatography, 125 mg is obtained. these "IL-3, 7a-bis-isopropylsonyloxy-1, 3.5 / 10 / estratrien-17A-ol.

PRI me R 11. a) A solution of 350 mg of 3, 7 o / -diacetoxy-17oZ-ethynyl-1, 3, 5/10 / -estratriene-17ft-ol in 20 ml of absolute benzene is mixed with 20 mg of n / toluenesulfonic acid and 1 ml of dihydropyran. Stir overnight at room temperature, dilute with ether, rinse until neutral with sodium bicarbonate solution and water and evaporate. 300 mg of 3, 7o 1-diacetoxy17o-ethynyl-17/3 -tetrahydropyranyloxy-1, 3.5 / 10 / -estratriene are obtained.

b) 250 mg of 3, 7oi, -diethoxy-17ol ethynyl-17 / -tetrahydropyranyloxy, 3.5 / 10 / -estraTriene is dissolved in 10 ml of methanol, a solution of 200 mg of potassium carbonate in 2 ml of water is added and boiled in 1.5 hours under a nitrogen atmosphere. Then the mixture is poured into ice water and organic, the substance is extracted with ether. After washing with water, drying and evaporation, 180 mg of 17 o6-ethynyl-17 (J-tetrahydropyranyloxy-1, 3.5 / 10) -estratrien3, 7 oL-ethynyl are obtained.

c) To a solution of 230 mg of 17 "-ethyn-17 / -tetrahydropyranyloxy-1, 3.5 / 10 / -e.stratrien-3, 7o -diol in

5 ml of pyridine at cooling with ice and in the atmosphere of nitrogen are added 0.5 ml of methanesulfonic acid and stirred for 38 hours at about 4 ° C in a atmosphere of nitrogen. Then everything is poured into ice-water, the precipitate is filtered off and dissolved in. methylene chloride. After washing with water, drying and evaporation, 200 mg of 17 °. Ytinyl-3, 7of-bis-mesyloxy-17 tetrahydropyranyloxy-1, 3.5 / 10 / estratriene are obtained in the form of a crude product, which is further used without further purification.

EXAMPLE 12 A solution of 200 mg of 17o-ethynyl-3, Id-- bis-mesyloxy-17 tetrahydropyranyloxy-1, 3.5 / 10 / estratriene in 5 ml of methanol is mixed with 500 mg of oxalic acid, dissolved in 2 ml of water and boil for 1/2 h. The mixture is then introduced into ice-cold water and the organic matter is extracted with methylene chloride. After washing with water, drying and evaporation, 17 oi-ethynyl-3, 70-bis-mesyloxy-1, 3.5 (10) -estratriene-17 f ol are obtained.

PRI me R 13. 3 g of ethinyl. Estradiol is incubated analogously to example 1 absidia orchides / ATCC8990 / aspergillus luchu- / CBS / ensis

Rhizopus nigricans / ATCC 62260 / Pellicularia bilamentosa / ATCC / 13289 /

Phizopus oryzal / ATCC4858 / Rhizopus kazanensis / ATCC 8998 / Rhizopus cohuu / ATCC 8998 /

or

Rhizopus stoloniber

Claims (2)

(ATCC 10404). After 45 hours of contact, the contents of the fermenter are treated twice with 10 liters of methyl isobutyl ketone and the extract is evaporated in a vacuum at a water bath at its temperature. The residue is purified in the same manner as 1 and recrystallized. 17o-eGinyl-1, 3.5 / 10 / estratriene-3, 7oi, 17/3-triol, which is identical to the product obtained in Example 1, is obtained. Claim 1. Invention 7ot-OR2. estr of diols of the general formula I OBj 4 --- tisCH is hydrogen, alkyl, where R. is a C-Cx-acyl, cyclopentyl or tetrahydropyranyl group; R2 - Ra is the same or different hydrogen, Cjf-C acyl or tetrahydro-uranyl group, characterized by the fact that .17 o6-ethynestrate; iol of general formula 11 HE-CHCH where R, has the indicated values, is fermented by a fungal culture of the genus absidia orchidis , aspergillus luchu- ensis, Rhizopus niqricans, P ellicularia Filamentoza, Rhizopus dryzal, Rhizopus kazanensis, Rhizopus cohuii, Rhizopus stolonifer or diplodia na-talensis in a concentration of 50-500 mg of the substrate for 50-500 mg of the substrate for a factor of 50–500 mg of the substratum, for the treatment of the body, for the application of the body, I will apply to the use of the body, I will apply to the volume at 25-35 s and, depending on the required value of R, R, and Rj in the final product, one or more hydroxyl groups are esterified to ethers or to esters, followed by separation of the target products:. . 2. The method according to claim 1, wherein the cyclopentyl halide in the presence of a base or dihydropyran in the presence of an acid is used to produce ethers. 3. The method according to claim 1, which is also characterized by the fact that anhydrides or acid chlorides are used to prepare esters in the presence of catalysts. Sources of information taken into account in the examination 1. US patent number 3318928, Cl. 260-397.5, pub. 09.05.67. 2. US patent number 3642992, cl. 424238, published. .72. Priorities for orders: 10/14/744 with E2I C-C-acyl residue of carboxylic acid. 08.08.75 when R2.H R - C-C-acyl residue of sulphonic acid.